The human immunodeficiency virus (HIV) encodes a nuclear transcriptional activator, Tat, which acts to enhance the processivity of RNA polymerase II (RNAPII) complexes that would otherwise terminate transcription prematurely at random locations downstream of the viral RNA start site. The mechanism of Tat transactivation is unique in that the cis-acting transactivation response element (TAR) is a stable RNA stem-loop structure that forms at the 5' end of nascent viral transcripts. Transcriptional activation by Tat through TAR requires proper folding of the RNA as well as specific bases in the bulge and apical loop of the TAR RNA hairpin structure (for review, see Cullen, B. (1993) Cell 73:417-420; Jones and Peterlin (1994) Annu Rev Biochem 63:717-743).
The interaction of Tat with TAR RNA is mediated through an arginine-rich motif (ARM) that is characteristic of a family of sequence-specific RNA-binding proteins (Gait and Karn (1993) Trends Biochem Sci 18:255-259). However, several lines of evidence suggest that the ARM of Tat is not an independent domain. First, the transactivation domain of Tat cannot be substituted by the activation domains of other transcription factors, such as the herpes virus VP16 protein, even though the VP16 activation domain is capable of activating transcription when tethered to RNA through a different RNA-binding domain (Tiley et al. (1992) Genes Dev 6:2077-2087; Ghosh et al. (1993) J Mol Biol 234:610-619). Second, the full-length Tat-1 protein, but not a mutant Tat protein that retains the ARM but lacks the transactivation domain, is able to target a heterologous protein to TAR RNA in vivo (Luo et al. (1993) J Virol 67:5617-5622), indicating that the activation domain is required to target Tat to TAR in the cell. Third, amino acid insertions that separate the Tat activation domain from the ARM strongly reduce transactivation through TAR, but do not affect TAR-independent transactivation by chimeric Tat proteins (Luo and Peterlin (1993) J Virol 67:3441-3445). Fourth, over-expression of mutant Tat proteins that contain the ARM does not block transactivation by the wild-type Tat protein in vivo (Madore and Cullen (1993) J Virol 67:3703-3711). In addition, residues in the core of the transactivation domain have been found to enhance the affinity and specificity of the Tat:TAR interaction in vitro (Churcher et al. (1993) J Mol Biol 230:90-110). Taken together, these studies strongly suggest that amino acid residues within the transactivation domain are required, directly or indirectly, for efficient binding of Tat to TAR RNA in vivo.
Tat recognizes a specific sequence in TAR that forms between the bulge and the upper stem, but does not require sequences in the loop of the hairpin that are essential for transactivation both in vivo and in vitro (for review, see Gait and Karn (1993) Trends Biochem Sci 18:255-259). Based on these findings, it has been postulated that Tat must interact with a host cell RNA-binding cofactor in order to recognize TAR RNA with high affinity and in a sequence-appropriate manner. Consistent with this possibility, it has been shown that high levels of Tat cannot overcome the specific inhibition of transactivation that occurs when cells are exposed to high levels of exogenous synthetic TAR "decoy" RNAs (Sullenger et al. (1990) Cell 63:601-608, Sullenger et al. (1991) J Virol 65:6811-6816; Bohjanen et al. (1996) Nucl Acids Res 24:3733-3738). Thus exogenous TAR RNAs appear to sequester a cellular cofactor in addition to Tat. Moreover, genetic studies indicate that a species-specific host cell factor is necessary for Tat to activate transcription through TAR in vivo. In particular, it has been found that murine and Chinese hamster ovary (CHO) cell lines do not support efficient transcription by Tat through TAR RNA (Hart et al. (1989) Science 246:488-491; Newstein et al. (1990) J Virol 64:4565-4567), whereas these same cell lines can support TAR-independent transactivation by chimeric Tat proteins (e.g., GAL4-Tat, Rev-Tat, MS2CP-Tat) that are targeted to their responsive promoters through a heterologous DNA- or RNA-binding domain (Alonso et al. (1992) J Virol 66:4617-4621; Newstein et al. (1993) Virol 197:825-828). Therefore the defect in nonpermissive rodent cells appears to be due to a problem of TAR RNA recognition.
Analysis of human:CHO hybrid cell clones reveals that a factor encoded on human chromosome 12 (Chr 12) can support a modest level of Tat activity in rodent cells (Hart et al. (1989) Science 246:488-491; Newstein et al. (1990) J Virol 64:4565-4567), and, most importantly, that the chromosome 12-encoded factor confers a specific requirement for sequences in the loop of TAR RNA that are otherwise dispensable for the residual low-level Tat activity that is observed in rodent cells (Alonso et al. (1994) J Virol 66:6505-6513; Hart et al. (1993) J Virol 67:5020-5024; Sutton et al. (1995) Virol 206:690-694). UV cross-linking studies have identified a cellular 83 kDa RNA-binding protein that is present in human and CHO-Chr12 cells, but not in CHO cells, which binds to TAR RNA in a loop-dependent manner (Hart et al. (1995) J Virol 69:6593-6599). Taken together, these results suggest that a human species-specific factor mediates the high-affinity, loop-specific binding of Tat to TAR RNA in vivo.
It has been generally presumed that the TAR RNA-binding cofactor would be distinct from the transcriptional coactivator for Tat. By contrast with the ARM, the N-terminal half of Tat can function autonomously as a transcriptional activation domain when fused to the DNA- or RNA-binding domain of a heterologous protein and targeted to an appropriate promoter. Truncated Tat-1 proteins that contain only the transactivation domain (aa 1-48) also act as potent dominant negative inhibitors of the wild-type HIV-1, HIV-2 and EIAV (equine infectious anemia virus) Tat proteins, suggesting that this region of Tat can sequester a limiting host cell transcription factor(s) that is necessary for Tat transactivation. Tat controls an early step in transcription elongation that is sensitive to inhibition by protein kinase inhibitors such as 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) (Kao et al. (1987) Nature 330:489-493; Laspia et al. (1993) J Mol Biol 232:732-746; Marciniak et al. (1990) Cell 63:791-802; Marciniak and Sharp (1991) EMBO J 10:4189-4196), and Tat transactivation in vivo and in vitro requires the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Chun and Jeang (1996) J Biol Chem 271:27888-27894; Okamoto et al. (1996)Proc Natl Acad Sci USA 93:11575-11579; Parada and Roeder (1996) Nature 384:375-378; Yang et al. (1996) J Virol 70:4576-4584).
The RNAPII carboxyl-terminal domain is predominantly unphosphorylated in assembled RNAPII preinitiation complexes and in complexes that pause shortly after initiation, but becomes heavily phosphorylated upon entry into productive elongation (for review, see Dahmus, M. (1996) J Biol Chem 271:19009-19012). Although the carboxyl-terminal domain is critical for gene expression in vivo and for regulated transcription in crude extracts, it is not required for basal promoter activity in purified reconstituted transcription systems (Serizawa et al. (1993) Nature 363:371-374). For many genes, the carboxyl-terminal domain has been found to be significantly more important for elongation than for initiation, which provides further support for the notion that carboxyl-terminal domain hyperphosphorylation may be an important step marking the transition of RNAPII molecules to forms that are competent for elongation (for review see Maldonado and Reinberg (1995) Curr Opin Cell Biol 7:352-361; Shilatifard et al. (1997) Curr Opin Genet Dev 7:199-204). The available evidence indicates that Tat acts through TAR RNA to regulate this DRB-sensitive, carboxyl-terminal domain kinase-dependent step in early elongation at the HIV-1 promoter (for review, see Jones, K A (1997) Genes Dev 11:2593-2599).
The possibility that Tat might interface directly with a carboxyl-terminal domain kinase was first suggested by the finding that both HIV-1 and HIV-2 Tat proteins interact very strongly, in vitro and in vivo, with a nuclear protein kinase (Herrmann and Rice (1993) Virol 197:601-608). Highly enriched fractions of the Tat-associated kinase (TAK) were shown to support hyperphosphorylation of the RNAPII carboxyl-terminal domain in vitro (Herrmann and Rice (1995) J Virol 69:1612-1620). Recently, the catalytic subunit of TAK was shown to be identical to a protein kinase subunit of P-TEFb, a RNAPII positive-acting transcription elongation factor complex that was purified originally from Drosophila cell transcription extracts (Mancebo et al. (1997) Genes Dev 11:2633-2644; Zhu et al. (1997) Genes Dev 11:2622-2632). Sequence analysis of the catalytic subunit of Drosophila P-TEFb establish its near identity to the human CDC2-related kinase, PITALRE, and the PITALRE kinase (hereafter called CDK9) has been shown to be critical for both TAK and P-TEFb activity (Mancebo et al. (1997) Genes Dev 11:2633-2644; Yang et al. (1997) Proc Natl Acad Sci USA 94:12331-12336; Zhu et al. (1997) Genes Dev 11:2622-2632). Immunoprecipitation of CDK9 from HeLa nuclear extracts effectively inhibits transcription elongation in vitro, and the residual transcription that remains is no longer sensitive to inhibition by DRB (Zhu et al. (1997) Genes Dev 11:2622-2632).
Independent support for a role for CDK9 in Tat transactivation comes from random drug screens for specific inhibitors of Tat, which yield novel compounds directed against the active site of CDK9 (Mancebo et al. (1997) Genes Dev 11:2633-2644), and the demonstration that a dominant negative mutant CDK9 protein blocks Tat activity in vivo (Mancebo et al. (1997) Genes Dev 11:2633-2644; Yang et al. (1997) Proc Natl Acad Sci USA 94:12331-12336). Interestingly, purified Drosophila P-TEFb is able to restore general transcription elongation to HeLa extracts that have been depleted of CDK9, but is unable to restore transactivation by Tat (Mancebo et al. (1997) Genes Dev 11:2633-2644; Zhu et al. (1997) Genes Dev 11:2622-2632), indicating that human-specific proteins associated with CDK9 may be necessary for Tat activity.
The human immunodeficiency virus (HIV-1) Tat protein is a potent activator of HIV-1 transcription that functions at an early step in elongation. Accordingly, there is a need in the art for a further understanding of the interaction(s) between the various components involved in Tat transactivation. A clearer understanding of these processes will facilitate the development of methods to modulate Tat transactivation, as well as assays for the identification of compounds useful for such modulation. These and other needs in the art are addressed by the present invention.